Due to the inability of adult cardiac myocytes to proliferate, spontaneous repair or regeneration of heart muscle is not normally possible. Consequently, pathological events such as myocardial infarction result in permanent damage that often leads to heart failure and death. As a potential therapeutic strategy, stem cells offer great potential due to their ability to differentiate into tissue-specific cell types guided by cues within the local niche microenvironment, possibly providing a cell source for cardiac repair. However, complications including low cell retention and viability, combined with a demanding and evolving post-infarction environment, have impeded the identification of mechanisms governing stem cell based treatments for myocardial infarction, resulting in failed clinical trials. Engineered cardiac tissues offer alternative experimental model systems combining a more physiologic 3D environment than the standard Petri dish, with long-term viability and improved experimental control not possible with natural heart muscle preparations. However, engineered cardiac tissues have been developed primarily for surgical repair applications, rather than as living in vitro systems designed for investigating myocardial injury, repair, and regeneration. This proposal aims to develop innovative new tools and approaches combining soft lithography and tissue engineering, with the overall objective of improving the understanding and efficacy of stem cell based approaches for cardiac repair. A guiding hypothesis is that mechanics of the 3D microenvironment is a key factor governing the interaction between human mesenchymal stem cells (MSC) and cardiac myocytes (CM). Two specific aims are designed to establish this new model system, test this hypothesis, and provide a springboard for future studies in this area. Aim 1: To develop a high-throughput engineered cardiac tissue (ECT) array system to evaluate and optimize stem cell co-culture strategies for enhancing cardiomyocyte contractile function in a controlled 3D microenvironment. This aim will focus on studying the effects of passive stretch, mechanical stiffness, and resident cardiac cell types on the ability of MSC to improve the contractile function of engineered cardiac tissues using a unique modular soft lithography based force sensor array. Aim 2: To establish a tissue engineered 3D cryo-infarct model to examine the effects of focal cell injury on stem cell migration, differentiation, and cardiac repair. This aim will combine the above engineered cardiac tissue array system with a novel cryo-infarct approach for examining MSC function in a controlled 3D model injury environment, helping to translate the findings of Aim 1 to experimental animal models of myocardial infarction or ECT implantation. Success of this high- risk, high-yield proposal should lead to new advances in understanding stem cell mechanobiology, facilitating translation to more complex experimental animal and clinical settings. PUBLIC HEALTH RELEVANCE: Because spontaneous repair or regeneration of heart muscle is not normally possible, pathological events such as myocardial infarction result in permanent damage that often leads to heart failure and death. Although stem cells offer great potential for cardiac repair, our understanding of the mechanisms guiding differentiation of stem cells is hampered by a lack of well-controlled experimental models of myocardial infarction that allow long term study of injury and repair processes. This proposal aims to develop innovative new tools and approaches combining soft lithography and tissue engineering, with the overall objective of understanding and directing stem cell differentiation for cardiac repair applications, which will hopefully lead to new advances in understanding stem cell mechanobiology and facilitate translation to more complex experimental animals and human patients.